Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or...
Reexamination Certificate
2000-03-23
2001-10-16
Schwartzman, Robert A. (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
C435S375000
Reexamination Certificate
active
06303289
ABSTRACT:
FIELD OF THE INVENTION
The present invention is directed to compounds and methods for the treatment of cancer and viral diseases, and is more particularly directed to the role of telomerase or inhibitors of purine synthesis and telomere structures as they relate to purine nucleotide synthesis and analogs and inhibitors of telomerase for the treatment of cancer and viral infections, including AIDS, and also to the use of inhibitors of interactions between telomerase and telomeres and viral proteins which are similar to those that participate in purine synthesis.
BACKGROUND OF THE INVENTION
Purines are critical biological molecules. They act as the precursors for DNA and RNA synthesis, as major components of cellular energy metabolism, as intracellular second messenger signaling molecules, as extracellular neurotransmitters, and as coenzymes for many critical biochemical reactions. All free-living organisms can synthesize purines. The de novo biosynthetic pathway consists of 10 enzymatic steps resulting in the synthesis of IMP. As might be expected, cancer cells, with their increased requirements for DNA and RNA synthesis, generally have elevated rates of purine synthesis. Therefore, it is not surprising that analogues of purines or molecules that inhibit purine synthesis are common anti-cancer drugs.
Viral infection has long been associated with human cancers. One of the first, and strongest, associations is with Epstein-Barr Virus (EBV), which is an etiologic agent in Burkitt's Lymphoma (BL), Hodgkin's disease, some T-cell lymphomas and nasopharyngeal carcinoma. EBV is a DNA virus that is related to the herpes viruses, and the most recent nomenclature recommendation is that it is renamed human herpes virus 4 (HHV4) (3). Recently, human herpes virus 8 (HHV8) has been strongly implicated as the etiologic agent in Kaposi's sarcoma in AIDS patents. The mechanisms by which EBV viral infection may work are still being clarified. Since viruses put heavy demands on the host DNA and RNA synthesis machinery, it may well be that during viral infection there is an elevated requirement for purine synthesis. Specifically, viruses may alter or assume control of cellular purine metabolism as a part of viral infection. As with cancer, some of the most effective antiviral agents for example, acyclovir, are purine nucleotide analogues. Interestingly, EBV and other viruses appear to possess genes important for nucleotide metabolism including ribonucleotide reductase, dihydrofolate reductase, and thymidine kinase. So far, no reports of viral genes participating directly in purine synthesis have been published.
SUMMARY OF THE INVENTION
The present inventors have observed an unexpected striking homology between an Epstein-Barr Virus (EBV) protein, BNRF
1
(Epstein Barr virus: X67777 or gi:59165), and a critical enzyme of purine biosynthesis, phosphoribosylformylglycinamide (FGAR), amido transferase (FGARAT)(Note: human AB002359 or gi:2224662; FGARAT
Drosophila melanogaster
is: U00683 or gi:414422). Another observation is that FGARAT or a related protein, (e.g., BNRF1), interacts with telomerase, the enzyme responsible for maintenance of telomeres, the ends of chromosomes. One aspect of the present invention, therefore, is directed to the regulation of various enzymes, cofactors and inhibitors that are involved in the stabilization of telomeres, and particularly inhibitors of a protein interaction with telomerase. The present inventors are the first to recognize that a protein encoded by various viruses, such as an EBV virus, is capable of carrying out one of the steps of purine biosynthesis, FGARAT. Because FGARAT interacts with telomerase, which acts to stabilize chromosome ends and is necessary for immortal growth of mammalian cells, the present inventors are the first to recognize that purine synthesis is directly linked to the immortalization process, a critical aspect of malignant transformation. This linkage occurs both because of the necessity for purine synthesis and also because of the interaction by particular proteins with telomerase.
Experiments indicate that viral FGARAT-protein BNRF1 has FGARAT enzymatic activity. Further, BNRF1, FGARAT and a second amino transferase in purine synthesis, amido-phosphoribosyltransferase (PRAT) (NM 002703 or gi:4505978), a rate-limiting step of purine synthesis in cells, also display enzymatic activity having an effect on telomerase function. Thus, another aspect of the present invention relates to the interaction of BNRF1, FGARAT and/or PRAT (hereinafter referred to alternatively as BNRF1-like proteins and/or proteins of the present invention) with telomerase, both in vitro and in vivo. By interfering with the interaction between BNRF
1
-like proteins and telomerase, one of skill in the art can for the first time regulate immortalization of a cell.
A relatively recent and exciting line of cancer research involves the study of telomerase. Chromosomes of cells all have ends called telomeres. It now appears that telomeres, which are composed of specific DNA repeat sequences, tend to shorten as cells divide. When telomeres get too short, cell division may cease. Telomerase repairs telomeres and maintains them at a size sufficient for continued cell division. Telomerase consists of at least 2 components, a reverse transcriptase subunit and an RNA component. In addition, at least one telomerase-associated protein has been reported. One interesting feature of EBV infection is that it can result in immortalization of human B-lymphocytes, possibly through the stabilization of telomeres and telomerase activity. The mechanism by which the virus accomplishes this, however, has hitherto been a mystery. Recently it has been possible to immortalize human fibroblasts by introducing and expressing the reverse transcriptase subunit of telomerase in these cells. These immortalized fibroblasts lack many other features of transformed cells. The present inventors contend that expression of telomerase is necessary but is not sufficient for immortalization and carcinogenicity. Clearly, one of the consequences of immortalization and cell division will be a requirement for purine nucleotide synthesis.
With respect to de novo purine nucleotide synthesis in animals, the present inventors have isolated Chinese hamster ovary (CHO) cell mutants deficient in each of the 10 enzymatic steps leading to IMP, the first completed purine nucleotide, and regionally mapped all 6 of the genes encoding the enzymes carrying out these steps. Using the CHO mutants, the present inventors have cloned the genes for a number of these enzymes in functional form using complementation of CHO mutants with cDNA or genomic DNA fragments. Genetic complementation by somatic cell hybridization was used to define independent genes encoding the enzymes of the pathway and the amidophosphoribosyl transferase (PRAT) gene was cloned. The PRAT gene encodes the first committed step of purine synthesis, is allosterically regulated, and is hypothesized to be an important site of regulation of the pathway. The present inventors have used the CHO mutants, and mutants of
Drosophilia melanogaster,
to define the multifunctional nature of the proteins carrying out the enzymatic steps. Using such mutants, the present inventors developed unique biochemical assays for the intermediates in the pathway and for the activities of the enzymes of the pathway. The mutants serve as a convenient source of large amounts of radio-labeled intermediates to use in enzyme assays.
The fourth step of the purine biosynthetic pathway, FGARAT, catalyzes the glutamine dependent amidation of FGAR to FGAM. It is the only enzyme of purine synthesis for which no mammalian gene or cDNA has been reported in the literature. CHO cell mutants of the AbeB complementation group lack detectable FGARAT activity and protein and have an absolute requirement for purines for growth. In addition, the present inventors have reported the isolation of an additional type of mutant, AdePAB, which is deficient in FGARAT activity and protein but is also d
Davis Katharine F
Elenor Roosevelt Institute
Schwartzman Robert A.
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